Immobilization Of Horseradish Peroxidase Via Click Chemistry And Its Direct Electrochemistry

Direct electron transfer between redox proteins and electrode has received considerable attentions in recent years. Electron transfer of redox proteins plays critical roles in many biological events, such as respiration and photosynthesis, and the mechanism of direct electron transfer from proteins to electrode surface and the corresponding electrocatalytic reactions can serve as models to understand the metabolic processes in biological systems. Moreover, the direct ET between redox proteins and contact is also of fundamental importance in life sciences and potential applications, for example, bioelectrocatalysis, bioelectronics, biosensors, and biofuel cells.Click chemistry is a modular approach that uses only the most practical and reliable chemical transformations. Its applications are increasingly found in all aspects of drug discovery, ranging from lead finding through combinatorial chemistry and target-templated in situ chemistry, to proteomics and DNA research, using bioconjugation reactions.It is a new kind of research field to apply click chemistry on the research of direct electron transfer of redox proteins, we try our best to combine click chemistry, self-assembled technology, carbon nanotubes, and electro-reduction of diazonium cations to study the direct electron transfer between grafting protein and electrode surface. The paper concludes following aspects:Chapter one:PerfaceBasic concept and characteristics of click chemistry are introduced, a number of click reaction types such as Huisgen 1,3-dipolar cycloaddtion, Diels-Alder reaction, Thiol-ene click reaction, and metal-free [3+2] cycloaddition reaction are summarized. Since click chemistry has been proposed, the application in drug discovery, polymer science, bioconjugate chemistry and functionalization of solid surface are more and more widely, and the applications in the above fields are highlighted.Charpter two:Direct electronchemistry of horseradish peroxidase immobilized on alkyl self-assembled monolayers via click chemistry A simple and versatile approach for covalent immobilization of redox protein on solid surface via self-assembled technique and click chemistry is reported. The alkynyl-terminated monolayers are obtained by self-assembled technique, then, azido-horseradish peroxidase (N3-HRP) was covalent immobilized onto the formed monolayers by click reaction. The modified process is characterized by reflection absorption infrared spectroscopy (RAIR), surface-enhanced Raman scattering spectroscopy (SERS) and electrochemical methods. All the experimental results suggest that HRP is immobilized onto the electrode surface successfully without denaturation. Furthermore, the immobilized HRP shows electrocatalytic reduction for H2O2, and the linear range is from 5.0 to 700.0μM. The heterogeneous electron transfer rate constant ks is 1.11 s-1 and the apparent Michaelis-Menten constant is calculated to be 0.196 mM.Charpter three:Direct electrochemistry of horseradish peroxidase immobilized on alkyl-MWCNTs via click chemistrySince carbon nanotubes have fast electron transfer rate and excellent biocompatibility, in this part, multi walled carbon nanotubes (MWCNTs) were utilized to promote the direct electron transfer between immobilized protein and electrode surface. First, gold electrode was modified with alkyl-MWCNTs via self-assembled technology, then, N3-HRP was covalent immobilized onto the MWCNTs by Huisgen 1,3-dipolar cycloaddtion reaction. The modified process was characterized by SERS, cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS), all the results indicated that immobilized HRP could achieve direct electron transfer with electrode surface, and shows excellent electrocatalytic reduction activity toward H2O2 with a linear range of 5.0×10-6 to 8.0×10-4M, the apparent Michaelis-Menten constant is calculated to be 0.653 mM.Charpter four:Direct electrochemistry of horseradish peroxidase immobilized on electrografted 4-ethynylphenyI film via click chemistryA simple two-step approach for the protein immobilization was introduced. First, alkynyl-terminated film was formed on electrode surface by electrochemical reduction of 4-ethylnylphenyl (4-EP) diazonium compound. Then, HRP modified with azido group was covalently immobilized onto the electrografted film via click reaction. RAIR and electrochemical methods (CV and EIS) were used to characterize the modification process. The results indicate HRP retains its native structure and fast direct electron transfer between electrode material and redox active proteins is achieved. Morover, the immobilized HRP shows excellent electrocatalytic reduction activity toward H2O2 with a linear range of 5.0×10-6 to 9.3×10-4 M.